Various oligonucleotides such as DNA and RNA have recently expanded their applications in the fields of treatment and diagnosis. Examples of the diagnostic application include DNA chip and DNA microarray. Examples of the therapeutic application include introduction of therapeutic gene and disease-related gene silencing by knockdown thereof. There are also attempts to use aptamers, which are nucleic acid or peptide molecules that specifically bind to a target molecule, as a therapeutic agent.
One of the especially interesting nucleic-acid technologies is a target gene knockdown technique by RNA interference (RNAi). RNAi is a process of silencing the activity of a gene by the action of a double-stranded RNA (dsRNA) molecule having the homologous sequence to the gene. In gene silencing by RNAi, the dsRNA molecule is recognized by Dicer which is one of the enzymes in the RNase III family and cut into small fragments called siRNAs (short interfering RNAs) of about 21 to 23 nucleotides long, an siRNA is incorporated into the RISC(RNA-induced silencing complex), and a homologous mRNA to the incorporated siRNA is cleaved at the center and degraded.
The knockdown technique however has problems of insufficient expression and difficulty in stable expression of an intended knockdown effect, because exogenous DNAs and RNAs are exposed to various nucleases in living organisms and RNAs are particularly sensitive to nuclease degradation.
To solve such a problem, chemical modifications on oligonucleotides have been studied for increasing nuclease resistance of oligonucleotides (Non-patent Documents 1 to 3). For example, an siRNA molecule has been attempted to be chemically modified with various substituents at a sugar, base, and/or phosphate moiety as shown in FIG. 1 (Non-patent Document 4).
In these circumstances, the present inventors have successfully introduced two moieties each having a benzene or pyridine structure at the 3′-end of a nucleotide using an amidite reagent for introducing the benzene or pyridine structure carried on CPG resin, as described in Patent Document 1 (see, e.g., FIGS. 2 and 3). This technique has been developed in consideration of a key role of the 3′-dangling end in RNAi as described below, and can enhance nuclease resistance of an oligonucleotide without decreasing knockdown effect thereof.
More specifically, of RISC, which is known as a multi-domain protein involving in a process of degradation of a target mRNA by RNAi, the PAZ domain has recently been subjected to X-ray crystallography in the form of cocrystal with siRNA (J. B. Ma., K. Ye and D. J. Patel., Nature, 429, 318-322 (2004).) The result showed that the PAZ domain recognized the 3′-dangling end of the siRNA through two nucleotide molecules of the 3′-dangling end slipping in a hydrophobic pocket of the PAZ domain.
(J. J. Song., J. Liu., N. H. Tolia., J. Schneiderman., S. K. Smith., R. A. Martienssen., G. J. Hannon and L. Joshua-Tor., Nat. Struct. Biol., 10, 1026-1032 (2003), K. S. Yan., S. Yan., A. Farooq., A. Han., L. Zeng and M. M. Zhou., Nature., 426, 468-474 (2003), Zhang., F. A. Kolb., L. Jaskiewicz., E. Westhof and W. Filipowicz., Cell., 118, 57-68 (2003), and A. Lingel., B. Simon., E. Izaurralde and M. Sattler., Nature., 426, 465-469 (2003)). From this finding, the present inventors thought that an oligonucleotide can exhibit an enhanced knockdown effect by chemical modification with two hydrophobic groups each having a benzene or pyridine structure as substitutes for two nucleotide molecules of the 3′-dangling end, and have developed the oligonucleotide derivative described in Patent Document 1.
In association with the present invention, the present inventors have also successfully developed a technique for enhancing nuclease resistance and activity of silencing of an siRNA by converting a diester phosphate bond of the 3′-dangling end of the siRNA into a carbamate or urea bond, or the negatively charged bond into a bond with no charge, and thereby providing better permeability through a nuclear membrane (Non-patent Document 5).